The overall goal of the following experiment is to quantify genomic instability in mouse B lymphocytes. This is achieved by first isolating the B cells for primary cell culture. Next, the cells are fixed facilitating the preparation of the metaphase spreads.
Finally, telomere PNA fish is performed for visualization of the fluorescently labeled telomeres. Ultimately, the level of various types of chromosome aberrations can be quantified by fluorescent microscopy. The implications of this technique can extend our understanding of the DNA repair pathways, as it can help identify genes that promote genomic stability in our targeted mouse models.
Though this method can provide insight into chromosome breaks and other large chromosomal rearrangements, it can also be adapted to measure other forms of genomic instability such as translocations. To isolate the B cells, begin by placing a freshly isolated spleen in a 35 millimeter tissue culture dish containing two milliliters of wash buffer in a laminar flow hood. Then use the backend of the plunger from a five milliliter syringe to gently macerate the spleen.
Next, filter the tissue slurry through a 70 micrometer nylon mesh strainer into a 50 milliliter tube using the plunger to gently press the bigger pieces of tissue through the strainer. Rinse the plate and syringe with eight milliliters of wash buffer, and filter the wash through the strainer. Then after spinning down the cells, pipette the pellet up and down a few times in three milliliters of a CK lysis buffer.
After five minutes, stop the reaction with 10 milliliters of wash buffer. Then after spinning down the cells again, tap the bottom of the tube to resuspend the pellet and add one milliliter of wash buffer to the cells. Next, incubate the cells with 50 microliters of anti CD 43 max micro beads on ice.
After 30 minutes, gently wash the cells in 10 milliliters of wash buffer. During the centrifugation place a minimax column on the magnet and a labeled 15 milliliter conical tube below the column. Then add one milliliter of wash buffer to the column, collecting the EIT in the tube.
Resuspend the cell pellet in one milliliter of wash buffer, and then load the cells onto the column After the sample has run through, rinse the column with one milliliter of wash buffer and add seven milliliters of wash buffer directly to the collected sample. After counting the collected cells, transfer them to a 50 milliliter tube for centrifugation. Then to activate the B cells, re suspend the pellet at one times 10 of the six cells per milliliter in supplemented B-cell medium and incubate the cells in six well plates at five milliliters of cells per well In a humidified cell culture incubator at 37 degrees Celsius and 5%carbon dioxide to fix the B cells.
Next at 50 microliters of smid to each of the wells. After a one hour incubation at 37 degrees Celsius, transfer the B cells to a labeled 15 milliliter tube. Cover the label with tape and spin down the cells.
Then aspirate all but about one milliliter of supernatant and resuspend the pellet by pipetting. Add five milliliters of 37 degrees Celsius, potassium chloride, drop by drop while pulsing on a vortex. Then add an additional 10 milliliters of potassium chloride and mix the cell solution by inversion.
Next, incubate the cells in a 37 degrees Celsius water bath for 15 minutes. Then add five drops of fresh fixative mixing once again by inverting. Then spin down the cells after resus suspending the pellet in one milliliter of supernatant, add five milliliters of fresh fixative, drop by drop while pulsing on a vortex and incubate the cells and an additional 10 milliliters of fixative at room temperature.
After 30 minutes, pellet the cells and remove all but one milliliter of the fixative. Then after washing the cells, two more times in 15 milliliters of fixative, re suspend the pellet in the final 70 microliters of the supernatant. Now place labeled slides at a 35 degree angle in a humidity chamber set to 22.9 degrees Celsius and 52%humidity.
Drop 35 microliters of the resuspended B cells onto each slide, preparing two slides per sample. Then after allowing the slides to dry in the humidity chamber for 30 minutes, store the slides in a slide box at 37 degrees Celsius until fully dry. Once dry, use a diamond pen to mark an 18 by 18 millimeter area for hybridization on the back of each slide to denature the slides for fish.
Next, apply 120 microliters of 70%deionized form amide to 24 millimeter by 60 millimeter cover slips, and then touch each slide to a cover slip. Denature the slides on an 80 degrees Celsius hot plate for 90 seconds, and then quickly and carefully slide off the cover slips and consecutively. Place the slides in ice cold 70, 90 and 100%Ethanol each for three minutes After the slides have dried, place the slides in a humid chamber and apply pre-need probe Mix to the marked areas on the slides.
Cover the probe mix with an 18 by 18 millimeter cover slip and incubate the slides at 37 degrees Celsius for one hour. Then wash the slides in pre-warned two XSSC with shaking. After five minutes, apply 35 microliters of moyal mounting medium to the slides.
Cover the medium with 24 millimeter by 60 millimeter cover slips and store the slides at four degrees Celsius. Finally, analyze the metaphase slides using a standard epi fluorescence microscope. Metaphase chromosomes derived from one mouse cell should form a discreet cluster containing 40 chromosomes.
Each chromosome has a centromere at one end, close to two telomere signals, as well as two additional telomere signals at the other end of the chromosome. Chromatid breaks are breaks in one of the two sister chromatids that make up each metaphase chromosome. In some cases, the broken end of the chromosome can be observed as a fragment with telomere signals in the same metaphase spread.
Radio chromosomes are complex rearrangements involving two chromosomes. Chromosomes may also become joined to form dicentric chromosomes, which appear as end-to-end fusions of chromosomes with centromeres at both ends. Robertsonian translocations are a type of translocation between the centromeres of two chromosomes, which appear as four sister chromat arms attached to a single centromere.
In this last image, a metaphase that does not have telomere signals present is shown. An absence of telomere signals can occur from errors in the probe preparation or from air bubbles under the cover slip during hybridization. Following this procedure, other methods like cell cycle analysis and colon information can be performed to answer additional questions like whether increased genomic instability affects cell viability.
